Difference gel electrophoresis based on lys/cys tagging.

Before separation, proteins of different biological samples are labeled with different fluorescent dyes, the CyDye DIGE Fluors. Currently three dyes with spectrally different excitation and emission wavelengths are available. This allows labeling up to three different samples, and coseparating them in one gel. The dyes can either be attached to the epsilon-amino side group of the lysine without derivatization of the polypeptides or to the cysteines after reduction of the disulfide bonds. For lysine labeling a so called minimal labeling approach is performed: only a low-ratio dye: protein is applied in order to prevent multiple labels per protein. Although only 3% of the proteins are tagged, the sensitivity of detection is comparable with the sensitivity of a good quality silver staining. The dyes are matched for size and charge to obtain migration of differently labeled identical proteins to the same spot positions. The spot pattern achieved with minimal labeling is similar to the pattern obtained with poststained gels. When cysteine tagging is applied, all cysteine moieties are labeled. This modification of the method affords extraordinarily high sensitivity of detection. However, because of multiple labeling, the resulting pattern will look different from nonlabeled or minimal labeled samples. The labeled samples are mixed together before they are applied on the gel of the first dimension. After separation the gels are scanned with the multifluorescent imager at the different wavelengths. Up to three images of comigrated protein mixtures are compared and evaluated from each gel. This multiplexing technique allows the application of an internal standard for each protein in a complex mixture: One of the labels is applied on a mixture of the pooled aliquots of all samples of an experiment. By coseparating this mixture with each gel an internal standard is created for reliable and reproducible detection and assessment of changes of protein expression levels. Image analysis is performed with special software, which allows codetection of protein spots across the different samples and the internal standard.